Effects of Stretch on the Bladder Umbrella Cell Apical Junctional Complex

Abstract

The urothelium, which lines the luminal surface of the bladder, must maintain its integrity during cycles of filling and voiding. How this is accomplished is not well understood. A critical component of the epithelial barrier is the apical junctional complex (AJC), which is a ring‐like structure circumscribing polarized epithelial cells at their apical pole. The AJC includes the apical‐most tight junction (TJ), subjacent adherens junction (AJ), and associated actin cytoskeleton, which modulates cell tension at regions of cell‐cell contact. We previously reported that the TJ ring encircling the outermost umbrella cell layer of the urothelium expands during bladder filling and contracts during voiding. However, whether the AJ is similarly affected, and the identity of the underlying machinery that drives these changes in the AJC remains unknown.When we examined bladders that were filled and voided in situ we observed the following: 1) Like the TJ ring, the AJ ring also expanded during filling and contracted within minutes of voiding. 2) In addition to actin, which was concentrated in a continuous ring interposed between the TJ and AJ, the actin bundling protein a‐actinin‐4, and non‐muscle myosin IIA, which regulates AJ dynamics, also associated with the AJC ring. 3) The actin disrupting agent cytochalasin D, as well as inhibitors of formins (SMIFH2) or Arp2/3 (CK869), decreased expansion of the AJC ring indicating a role for active actin polymerization in this process. Surprisingly, treatment with blebbistatin, a myosin II contraction inhibitor, had no effect on the expansion of the AJC ring. 4) An exocytosis inhibitor, brefeldin A, showed a strong inhibitory effect on AJC ring expansion, indicating that vesicular traffic may regulate AJC dynamics. 5) To target the exocytic machinery more specifically we expressed dominant‐negative (DN) RAB8A, RAB11A, or RAB13 mutants. Whereas DN‐RAB13, previously implicated in the regulation of TJ dynamics, significantly decreased AJC ring expansion during bladder filling, DN‐RAB8A and DN‐RAB11A, which are involved in stretch‐stimulated exocytosis during bladder filling, did not. 6) Unlike expansion of the AJC ring, its voiding‐induced contraction was strongly inhibited by blebbistatin and was also significantly inhibited by cytochalasin D. 7) Intriguingly, AJC ring contraction was also inhibited by the general endocytosis inhibitor Pitstop2 or expression of a DN mutant of the endocytic regulatory protein Dynamin2. Moreover, DN‐RhoA, which we previously implicated in umbrella cell apical endocytosis, similarly impaired AJC ring contraction.ConclusionsOur studies indicate that a likely mechanism for retention of urothelial continuity in the face of cyclical changes in volume is expansion and contraction of the AJC ring. Not only does AJC ring expansion depend on actin polymerization, it also requires exocytosis, presumably of vesicles containing AJC‐associated membrane proteins. In contrast, contraction of the AJC ring is likely mediated by actomyosin contraction of the junctional ring and endocytosis of excess AJC proteins.Support or Funding Information1F31DK117547‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.